This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To define the influence of specific extracellular matrix components and placental mesenchymal cells on trophoblast differentiation from human embryonic stem cells. Our laboratory has established an in vitro model to study trophoblast differentiation. Human embryonic stem cells (hESC) will consistently differentiate to trophoblasts and secrete high levels of placental hormones when allowed to form embryoid bodies (EBs), and are transferred into a three-dimensional (3D) extracellular matrix (Matrigel) environment (ECM). Hormone secretion was enhanced in this 3D system in comparison with planar trophoblast outgrowths in standard adherent culture. The invasion of extracellular matrix and the uterine endometrium by extravillous trophoblasts is a crucial component of pregnancy success in human and nonhuman primates. To define the invasive characteristics of the hESC-derived extravillous trophoblasts, we worked to determine optimal systems for monitoring and quantifying trophoblast migration. We have determined that embryoid bodies derived from aggregates of approximately 500 hESC produce maximal levels of hCG, a marker of trophoblast differentiation, in comparison with larger embryoid bodies. Studies of migration with embryoid bodies stained with a lipophilic fluorescent dye proved unsatisfactory due to high background fluorescence in the migration chambers employed. Modifications have been introduced which provide better control over fluorescence, quantitation and migration. These changes include enrichment of embryoid body trophoblasts by selection following outgrowth on 2-dimensional Matrigel surfaces, and labeling with Quantum Dots fluorescent nanoparticles for quantification of migration by confocal microscopy. We conclude that the migration of hESC-derived trophoblasts, and its modulation by specific culture surfaces, can be quantitatively monitored with these methods. This work used federally approved hES cell lines.